Oropharyngeal candidiasis (OPC, thrush) is the term given to opportunistic oral infection caused by the yeast Candida, and is the most common mycotic complication associated with human immunodeficiency virus (HlV)-infected patients. The ability of Candida isolates to form biofilm on devices and host tissue surfaces is believed to be intricately linked with OPC infections and denture stomatitis. Therefore, understanding the role of biofilms in pathogenesis and host-Cand/cfa interactions is critical. In this application, to gain insight into these interactions, we will use a proteomics-based approach to identify specific proteins that are central to the biofilm forming ability of C. albicans and determine their role in interactions between fungal biofilm and host tissues. We have previously established an in vitro model of C. albicans denture biofilm (Publication 1, Appendix). Using this model, we: 1) identified the developmental phases of candidal biofilm formation (Publication 1, Appendix), 2) investigated the antifungal resistance profile of C. albicans biofilms at different growth phases (Publication 2, Appendix), 3) investigated the multifactorial mechanisms of azole resistance of early and mature biofilms formed by C. albicans (Publication 3, Appendix), 4) used a proteomic approach to identify potential target proteins that are differentially expressed in early phase biofilms (see Publication 4, Appendix), 5) used molecular and biochemical methods to show that one of the identified proteins (alcohol dehydrogenase, Adhlp) is a negative regulator of Candida biofilm (Publication 4, Appendix), and 6) moved our studies closer to the clinical setting by using an engineered human oral mucosa (EHOM), developed recently by Dr. Rouabhia (Co-investigator on this application). This collaborative research between the applicant and Dr. Rouabhia showed that Adhlp plays an important role in both Candida biofilm formation and invasion of host mucosal tissues (Manuscript submitted, Appendix). The overall hypothesis of this application is that C. albicans express specific proteins that are essential for biofilm formation and play critical roles in Candida-host tissue interactions. We will test our hypothesis using the following Specific Aims: Aim 1. Identify early phase biofilm-specific proteins expressed by C. albicans. Aim 2. Determine whether the identified proteins are critical to the ability of C. albicans to form biofilms in vitro by disrupting genes encoding them. Aim 3. Use an in v/Vo-like Engineered Human Oral Mucosa (EHOM) Model to determine whether the identified proteins are essential for Candida biofilm formation and host tissue damage. Aim 4. Validate the in vitro and EHOM results in vivo using a murine oral model of Candida biofilms. Experiments described in this application will provide insight into the biology of OPC-associated C. albicans biofilms and may suggest potential therapeutic and diagnostic targets for the prevention, treatment, and diagnosis of oral Candida infections.